Nucleotide Identification and Orientation Discrimination of DNA Homopolymers Immobilized in a Protein Nanopore

Abstract

Shown below are the results from a comparison of fully methylated and unmethylated versions of the BRCA1 and MS3 genomic DNA fragments(A). Using quantitative PCR, we measured the number of DNA molecules which translocated through a 1.8nm nanopore at a given voltage(B). Unmethylated MS3 and BRCA1 translocate above 3.77V and 3.61V, respectively while the thresholds for fully methylated MS3 and BRCA1 are 2.53V and 2.69V respectively.

Microfluidic-based biosensors toward point-of-care detection of nucleic acids and proteins

“…Here, the natural nanopore a-Haemolysin showed that the diameter of its lumen is suitable for providing measurable differences in current upon DNA translocation ( Fig. 2) (Maglia et al 2008;Purnell et al 2008;Butler et al 2008;Purnell and Schmidt 2009). Nanopore-based sequencing methods are able to detect single DNA strands, and the signal can be distinguished from the background noise.…”

Charge, Diffusion, and Current Fluctuations of Single-Stranded DNA Trapped in an MspA Nanopore

“…The ssDNA oligonucleotides used in this work were purchased from Integrated DNA Technologies (Coralville, IA) with dual HPLC purification. The sequences used here are (dA) 27 , (dC) 27 , (dT) 27 , and (dGdA) 13 (dG), all with 3 0 biotinylation. The GA sequence was chosen as an alternative to homopolymer (dG) to avoid G-tetrad formation.…”

“…The GA sequence was chosen as an alternative to homopolymer (dG) to avoid G-tetrad formation. Even so, it seems that G self-association still caused some notable differences during the (dGdA) 13 (dG) measurement (see the Supporting Material). ssDNA-NeutrAvidin complexes were formed by adding a dilute solution of ssDNA to a concentrated solution of NeutrAvidin (Thermo Fisher Scientific, Waltham, MA) dropwise, with rapid stirring.…”

“…1 A illustrates how a charged ssDNA molecule with a molecular stop attached can be threaded through and trapped in a nanopore by applying a voltage bias across the pore. In previous work, molecular stops such as an enzyme (4)(5)(6)(7)(8), bound protein (9)(10)(11)(12)(13), or a DNA double strand (2,14,15) have been used to assist in DNA trapping. The magnitude of the ssDNAinduced ionic current blockage depends on the sequence of ssDNA nucleotides near the pore's constriction (4,8,16,17).…”

RNA and DNA Diagnostics

“…22) (Maglia et al 2008). The DNA sequences can even be identified by this way (Purnell et al 2008;Purnell and Schmidt 2009;Stoddart et al 2009;Butler et al 2008). Identification of nucleobases is also possible using conducting nanogaps inside nanochannels, able to measure the electrical conductivity perpendicular to the channel (Liang and Chou 2008).…”